Further improvement in lipid productivity of the strain was achieved by performing conventional mutagenesis and screening on the best producing strains. Further evaluation of the strain under different trophic (photoautotrophic and mixotrophic) modes showed significant variation in biomass productivity (73 to 114 mg L-1 day-1) and total lipid productivity (35.02 to 50.42 mg L-1 day 1).
Biochemical characterization of Chlorella sp. FC2 IITG under different trophic modes, nutritional starvation and outdoor
- Characterization of the strain in open pond under outdoor
- Analysis of growth, substrates utilization and biomass
- Quality assessment of biodiesel generated from FC2 under
- Evaluation of the strain Chlorella sp. FC2 IITG under
- Characterization of the strain in open pond under outdoor
- Composition of FAME obtained from Chlorella sp. FC2 IITG grown under different cultivation conditions 106
FC2 IITG grown under different cultivation conditions 106 IITG grown under different cultivation conditions 106 4.3.5 Quality assessment of biodiesel obtained from Chlorella.
5 Media engineering and process optimization for improved biomass titer of Chlorella sp. FC2 IITG 116
Effect of light intensity on growth and biomass
Analysis of growth, substrate utilization and FAME
High cell density biomass formation in fed-batch mode with constant and dynamic light intensity 148
7 Enhancement of lipid content in Chlorella sp. FC2 IITG
Analysis of growth, substrate utilization and enzyme
An enzyme assay to understand the carbon partitioning mechanism of the mutant and wild-type strain.
8 Flux balance analysis and enzyme activity assay in Chlorella
Development of the kinetic model for dynamic FBA
Dynamic flux balance analysis for light dark metabolism
L ist of Figures
Dynamic profiles for growth, changes in intracellular neutral lipid content of the strain Chlorella sp. Dynamic profiles for growth, neutral lipid accumulation and substrate utilization of the strain Chlorella sp.
L ist of Tables
ANOVA for the quadratic regression model obtained from CCD- RSM used in the optimization of media components for the. The light availability per cell was calculated assuming a constant light intensity of 250 µE m-2 s-1 and for the dynamic change in light intensity from 250 to 450.
Introduction
- Background and motivation
- Objectives of the study
- Approach
- Organization of the thesis
- References
Further improvement in the net lipid productivity of the strain was achieved by performing conventional mutagenesis and screening for the best mutant producers. Finally, Chapter 9 summarizes the current research with the conclusions drawn and the future prospects of the work.
Review of Literature
Energy fuel crisis and alternate renewable energy resources
Biomass-based energy generation has gained considerable interest as one of the potential alternatives due to the following facts (i) it can be used for the generation of various forms of biofuel: gaseous (hydrogen and methane), liquid (ethanol, methanol , butanol, biodiesel) , Fischer-Tropsch diesel) and solid fuels; (ii) it is renewable in nature and can be made sustainable in the future; (iii) high energy yield efficiency; (iv) reduced greenhouse gas emissions compared to other fuels; (v) sustainability in terms of net zero carbon emissions making the whole process carbon neutral and (vi) significant economic potential as it can cost low (Demirbas, 2008; Singh et al., 2010). However, all these sustainable technology developments are based on the raw material economy, its availability, production costs and conversion capabilities (Singh et al., 2010).
Biodiesel and available resources
However, with increasing demand, these food reserves were found insufficient to meet current energy requirements (Nautiyal et al., 2014). The advantages and disadvantages of different biodiesel feedstocks are discussed in Table 2.1 which highlights the advantages of microalgal systems over other feedstocks.
Microalgal biodiesel and characteristics
They accumulate carotenoids and polysaccharides which are used as therapeutics and bioplastics, respectively (Hempel et al., 2011). Therefore, microalgae are believed to be the only potential nutrients that can meet current energy requirements (Chisti, 2007; Mata et al., 2010).
Microalgae: classification and biology
These groups are further divided into several by secondary endosymbiosis (details taken and modified from Croft et al., 2006). Other members of the group arise from a recurrent endosymbiotic relationship that existed between these variants (Croft et al., 2006).
Biochemistry of microalgae
Presence or absence of nutrients in the growing media will influence the nature, quantity and quality of the product (Karemore et al., 2013). With an increase in temperature, the content of saturated fatty acids in the total fraction increases and with a decrease in temperature, the content of unsaturated fatty acids increases in concentration (Hu et al., 2008).
Culturing of microalgae: mode of nutrition and reactor types
- Mode of nutrition
- Various reactor systems used for algal culturing
Maximum net lipid productivity was reported for mixotrophic, followed by heterotrophic and photoautotrophic cultivation conditions (Wang et al., 2014a). Where 𝐼𝑑 represents the light intensity at depth d, 𝐼0 is the initial incident intensity, 𝛾 is the turbidity (Chen et al., 2011).
Processing of microalgal biomass for biodiesel generation: Current harvesting and conversion technologies
- Various dewatering methods used for algal harvesting
- Biomass to biodiesel conversion technologies
However, the use of ozone for flotation is not economically feasible for biodiesel production (Rawat et al., 2011). Spray drying, drum drying and sun drying are commonly used to remove the water content of algal biomass (Pragya et al., 2013).
Systems biology of microalgae
Another interesting discovery is the identification of the nitrogen response regulator NRR1 in Chlamydomonas reinhardtii which is induced under nitrogen starvation conditions and during neutral lipid accumulation (Boyle et al., 2012). Alternatively, the constraint-based metabolic models that can quantitatively predict the dynamic responses of the cells with respect to the changes in environmental conditions (Kauffman et al., 2003).
Current challenges
Although many genome-scale metabolic models have been constructed for microalgal systems, no model has highlighted the pathways or enzymes that must be altered to maximize net lipid productivity, and more research in this area may allow us to define the targets of susceptible to metabolic engineering towards biodiesel production.
Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS (2011) Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Lam MK, Lee KT (2012) Immobilization as a feasible method to facilitate the separation of microalgae from water for biodiesel production.
Isolation, screening, identification and characterization of microalgae for neutral lipid accumulation
Background and motivation
Biomass productivity and high lipid content of microalgae are therefore mutually exclusive, which in turn lowers the net lipid yield (Hu et al., 2008). To that end, it is indispensable to develop an efficient process with a suitable producer strain that aims at both optimal growth and lipid productivity (Hu et al., 2008).
Materials and methods
- Sampling and isolation of indigenous microalgal strains
- Selection of the growth medium for indigenous microalgal strains
- Screening and selection of neutral lipid accumulating microalgal strains
- Identification of the microalgal strain
- Characterization of the strain under different physico-chemical conditions All the characterization experiments were performed using 1 % (v/v) mid log phase
- Analytical techniques
For molecular analysis, the cells were disrupted and the genomic DNA of the strain was extracted using DNeasy Plant Mini Kit (Qiagen, Valencia, CA). The biomass formation was monitored by obtaining the dry cell weight of the algal cells.
Results and discussion
- Sampling and isolation of indigenous microalgal strains
- Selection of the growth medium for growth of indigenous microalgal strains The growth medium for ten different strains was determined by growing them in six
- Screening and selection of neutral lipid accumulating microalgal strains The main aim of screening is to identify the “Fat algae” which can accumulate
- Morphometric and molecular identification of the organism
- Effect of pH, temperature, carbon and nitrogen sources on growth of FC2 The strain was able to grow over a wide range of pH 4 to 10 and its optimal growth
Regardless of cultures, BG11 medium supported the growth of all strains than any other medium composition (Fig. 3.2 and 3.3). The neutral lipid content of the organism was screened based on Nile Red neutral lipid detection under confocal microscope as described in section 3.2.6.
Conclusions
Glucose was found to be the optimal carbon source for growth with the highest biomass titer, followed by sodium acetate and fructose. Mannitol (Kessler and Hus, 1992) and glycerol (Heredia-Arroyo et al., 2011) were found to inhibit the growth of most Chlorella species.
Chen W, Zhang CW, Song LR, Sommerfeld M, Hu Q (2009) A high throughput Nile-red method for quantitative measurement of neutral lipids in microalgae. Cooksey KE, Guckert JB, Williams SA, Callis PR (1987) Fluorometric determination of neutral lipid content of microalgal cells using Nile red.
Background and motivation
The isolate was evaluated for growth and lipid productivity under photoautotrophic and mixotrophic conditions. Finally, the culture was grown under outdoor conditions to evaluate its growth performance and lipid productivity under fluctuating environmental parameters and in the presence of pollutants.
Materials and Methods
- Evaluation of the strain under different trophic modes
- Characterization of the strain under nitrogen and phosphate starvation conditions
- Characterization of the strain in open pond under outdoor condition
- Analysis of growth, substrates utilization and biomass composition
- Analysis of fatty acids methyl esters (FAME) derived from microalgae
- Quality assessment of biodiesel generated from FC2 under different cultivation conditions
Nitrate estimation in the supernatant was performed using salicylic acid method with sodium nitrate as the standard (Cataldo et al., 1975). Dynamic profile of neutral lipid accumulation in the biomass was obtained by Nile Red-based assay method and the total intracellular lipid was measured as fatty acid methyl esters (FAME) along with fatty acid composition in gas chromatography (GC).
Results and Discussion
- Evaluation of the strain Chlorella sp. FC2 IITG under different trophic modes Biomass and lipid productivity of the organism FC2 was evaluated under
- Characterization of growth and lipid productivity of Chlorella sp. FC2 IITG under nitrate and phosphate starvation
- Characterization of the strain in open pond under outdoor condition
- Composition of FAME obtained from Chlorella sp. FC2 IITG grown under different cultivation conditions
- Quality assessment of biodiesel obtained from Chlorella sp. FC2 IITG
The biomass productivity of the strain under mixotrophic condition (73 mg L-1 day-1) was found to be less than photoautotrophic (114 mg L-1 day-1) condition (Table 4.1). Quality assessment of biodiesel obtained from strain FC2 grown under different cultivation conditions was performed through empirical equations as a function of FAME composition obtained experimentally (Su et al.
Conclusions
Huo S, Wang Z, Zhu S, Zhou W, Dong R, Yuan Z (2012) Cultivation of Chlorella zofingiensis in bench-scale outdoor ponds by adjusting pH using dairy wastewater in winter, South China. Feng P, Deng Z, Fan L, Hu Z (2012) Lipid accumulation and growth characteristics of Chlorella zofingiensis under different nitrate and phosphate concentrations.
Media engineering and process optimization for improved biomass titer of Chlorella sp. FC2 IITG
Background and motivation
Therefore, optimization of stoichiometrically limiting and limiting substrate concentrations in the medium remains an important task in bioprocess development. A two-fold increase in biomass concentration was achieved through optimization of ingredients in Bold Basal medium for the photoautotrophic growth of Chlorococcum infusionum (Karemore et al., 2013).
Materials and methods
- Cultivation conditions
- Media engineering for growth of FC2 .1 Design of experiments
- Process optimization for maximization of growth
- Analysis of growth, substrate utilization and FAME composition
The experimental data obtained from the CCD were fitted to the polynomial equation and the significance of the predicted model parameters was determined through Analysis of Variance (ANOVA) and statistical regression analysis. Further, the maximization of biomass productivity was achieved by changing the availability of light for the organism to the optimized composition of the environment.
Results and discussion
- Media engineering for growth of FC2 using statistical hybrid optimization tools Most of the media optimization studies utilize one at a time strategy to determine
- Effect of light intensity on growth and biomass productivity of FC2
Reactor level characterization of FC2 in optimized BG11 medium resulted in a biomass titer of 1.0 g L-1 under photoautotrophic reactor conditions as detailed in Section 4.2.1. Changes in light intensity significantly affected the growth of FC2, resulting in a maximum biomass titer of 5.66 g L-1 at 250 µE m-2 s-1.
Conclusions
Franco-Lara E, Link H, Weuster-Botz D (2006) Evaluation of artificial neural networks for modeling and optimization of media composition with a genetic algorithm. Wang J, Wan W (2009) Optimization of fermentative hydrogen production process using genetic algorithm based on neural network and response surface methodology.
Background and motivation
Lipid content and biomass productivity are two mutually exclusive traits and simultaneous improvement of both these parameters remains a challenge in algae-based biodiesel production (Hu et al., 2008). Different strategies have been proposed to improve the lipid content without affecting the biomass productivity under photoautotrophic conditions (Hu et al., 2008; Rodolfi et al., 2009).
Materials and methods
- Organism, media and growth conditions
- Experimentation
- Analysis of growth, substrate utilization and FAME composition
The first phase represents the growth phase in which culture of FC2 with high cell density was achieved via intermittent dosing of urea and phosphate together with dynamic. The urea concentration in the medium was estimated using the diacetyl monoxime method as prescribed by Wybenga et al.
Results and discussion
- High cell density biomass formation in fed-batch mode with constant and dynamic light intensity
- Lipid enrichment in FC2 via nitrogen starvation
The fed batch with dynamic increase in light intensity resulted in maximum biomass titers of 13.5 g L-1 and biomass productivity of 675 mg L-1 day-1 (Fig. 6.4A). Therefore, to achieve high cell density cultivation, dynamic increase in light intensity is required along with intermittent supply of the limiting nutrients.
Conclusions
Two unoptimized phases represent FC2 growth in unoptimized BG11 media at 20 µE m-2 s-1. No significant change in FAME compositions was observed compared to the unoptimized medium which supports the organism as a suitable candidate for biodiesel production.
Imaizumi Y, Nago N, Yusoff FM, Taguchi S, Toda T (2014) Estimation of optimum specific light intensity per cell on a high-cell-density continuous culture of Chlorella zofingiensis not limited by nutrients or CO2. Han F, Huang J, Li Y, Wang W, Wan M, Shen G, Wang J (2013) Improved lipid productivity of Chlorella pyrenoidosa by the culture strategy of semi-continuous cultivation with nitrogen limitation and pH control by CO2.
Background and motivation
In the present study, UV radiation was used to induce mutation in the wild type Chlorella sp. The distinct changes in the major lipid and carbohydrate biosynthetic pathways were also captured by key enzyme activity assays to understand the regulation of lipid biosynthesis in the mutant.
Materials and Methods
- Microalgae and cultivation conditions
- UV mutagenesis
- Screening of the UV mutants
- Evaluation of the mutant under photoautotrophic cultivation condition and two stage nitrogen starvation condition
- Analysis of growth, substrate utilization and enzyme activity
Suitable blanks were taken for individual samples consisting of all components together with enzyme extract in the absence of substrates (1, 2-Di Oleyl Glycerol and oleoyl-CoA). A standard curve of CoA-SH (ranging from 0 to 100 µM CoA-SH) was generated along with an assay to calculate the amount of CoA-SH produced in the DGAT reaction.
Results and discussion
- UV mutagenesis and screening of the UV mutants
- Evaluation of the mutant under photoautotrophic cultivation conditions .1 Evaluation under photoautotrophic batch growth condition
A similar increase in lipid content of up to 28% was observed in a Nannochloropsis HP-1 mutant strain obtained by heavy ion irradiation (Ma et al., 2013). A slow increase in chlorophyll content with increasing biomass titer was observed, and the highest chlorophyll content was reached on day 5 of cultivation in the mutant strain, while in the case of the wild-type strain.
